This application seeks support for an established research program in bioinorganic chemistry directed toward a molecular understanding of biologically relevant iron-sulfur clusters and related species. These clusters, particularly the Fe4S4 cubane-type, are of pervasive occurrence in all forms of life. In addition to their well-recognized role as essentially isolated electron transfer centers in proteins, these clusters are now known to be covalently bonded to catalytic sites and to act as catalytic sites themselves. These are aspects of a larger collection of structural and reactivity features localized at a specific iron subsite of a cluster. The proposed research takes new directions in the form of detailed investigations of subsite-specific and other properties of iron-sulfur clusters. These studies will be undertaken: (1) synthesis and Fe4S4 cluster insertion reactions of a new cyclic trithiol and its subsequent use in examination of subsite-specific properties; (2) delineation of the scope of subsite-specific reactions of Fe4S4 clusters; (3) characterization of the reactions of CO with subsite- differentiated, reduced clusters as one means of examining CO inhibition of Fe-S enzymes; (4) characterization of subsite-specific reactions of the substrate and inhibitors of aconitase; (5) formation and characterization of bridged Fe4S4-X assemblies (X - Fe4S4, Fe, Ni, and other metals) as potentially related to the P-clusters of nitrogenase and the catalytic units of nitrite and sulfite reductases and carbon monoxide dehydrogenases; (6) formation of the cuboidal Fe3S4 cluster, the latest Fe-S cluster to be found in proteins, by oxidation conversion of a subsite-differentiated Fe4S4 cluster or reductive isomerization of a linear Fe3S4 Cluster; (7) synthesis of heterometal cubane-type clusters MFe3S4 by insertion of biological metal M - Mn, Co, Ni, Cu, Zn into cuboidal Fe3S4 or by reductive incorporation in, with rearrangement of, linear Fe3S4; (8) accomplishment of the six-electron reductions SO3 2- -> S2- and NO2- -> NH3 at Fe(II) with emphasis on the identification of intermediate states of substrate reduction; (9) preparation and characterization of polynuclear cyclic and bicyclic clusters; (10) preparation and characterization of high-nuclearity Fe-S/Se clusters by means such as self-assembly and cluster enlargement, as a route to uncharacterized biological clusters such as the Hclusters of hydrogenase; (11) utilization of high-nuclearity clusters as a means to the preparation and characterization of V-Fe-S and Mo-Fe-S clusters that constitute the cofactors of V- and Mo-nitrogenase.